1.1 Field of the Invention
The present invention relates to plant genetics and enzymes. More specifically, it concerns methods for identifying nucleic acid segments encoding polypeptides having acetyl-CoA carboxylase (ACCase) activity, and methods for detecting ACCase polypeptides which are resistant to herbicides of the aryloxyphenoxypropionate or cyclohexanedione classes.
1.2 Description of the Related Art
1.2.1 Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the first committed step in de novo fatty acid biosynthesis, the addition of CO.sub.2 to acetyl-CoA to yield malonyl-CoA. It belongs to a group of carboxylases that use biotin as cofactor and bicarbonate as a source of the carboxyl group. ACCase catalyzes the addition of CO.sub.2 to acetyl-CoA to yield malonyl-CoA in two steps as shown below. EQU BCCP+ATP+HCO.sub.3.fwdarw.BCCP-CO.sub.2 +ADP+P.sub.i (1) EQU BCCP-CO.sub.2 +Acetyl-CoA.fwdarw.BCCP+malonyl-CoA (2)
First, biotin becomes carboxylated at the expense of ATP. The carboxyl group is then transferred to Ac-CoA (Knowles, 1989). This irreversible reaction is the committed step in fatty acid synthesis and is a target for multiple regulatory mechanisms. Reaction (1) is catalyzed by biotin carboxylase (BC); reaction (2) by transcarboxylase (TC); BCCP=biotin carboxyl carrier protein. In eukaryotic ACCase, all domains are located on one large polypeptide (e.g., animal, plant and yeast ACCase).
Yeast, rat, chicken and human ACCs are cytoplasmic enzymes consisting of 250- to 280-kDa subunits while diatom ACCase is most likely a chloroplast enzyme consisting of 230-kDa subunits. Their primary structure has been deduced from cDNA sequences (A1-feel et al., 1992; Lopez-Casillas et al., 1988; Takai et al., 1988; Roessler and Ohlrogge, 1993; Ha et al., 1994). In eukaryotes, homologs of the four bacterial genes are fused in the following order: accC, accB, accD and accA. Animal ACCase activity varies with the rate of fatty acid synthesis or energy requirements in different nutritional, hormonal and developmental states. In the rat, ACCase mRNA is transcribed using different promoters in different tissues and can be regulated by alternative splicing. The rat enzyme activity is also allosterically regulated by a number of metabolites and by reversible phosphorylation (Ha et al, 1994, Kim, 1997). The expression of the yeast gene was shown to be coordinated with phospholipid metabolism (Chirala, 1992; Haslacher et al., 1993).
While strong evolutionary conservation exists among biotin carboxylases and biotin carboxylase domains of all biotin-dependent carboxylases, BCCP domains show very little conservation outside the conserved sequence E(A/V)MKM (lysine residue is biotinylated) (Knowles, 1989; Samols et al, 1988). Although the three functional domains of the E. coli ACCase are located on separate polypeptides, plant ACCase is quite different, having all 3 domains on a single polypeptide.
At least one form of plant ACCase is located in plastids, the primary site of fatty acid synthesis. The gene encoding it, however, must be nuclear because no corresponding sequence has been seen in the complete chloroplast DNA sequences of tobacco, liverwort or rice. The idea that in some plants plastid ACCase consisted of several smaller subunits was revived by the discovery of an accD homolog in some chloroplast genomes (Li and Cronan, 1992). Indeed, it has been shown that the product of this gene in pea binds two other peptides, one of which is biotinylated. The complex may be a chloroplast isoform of ACCase in pea and some other plants (Sasaki et al., 1993).
It has been shown recently that plants have indeed more than one form of ACCase (reviewed in Sasaki et al., 1995, Konishi et al., 1996). The one located in plastids, the primary site of plant fatty acid synthesis, can be either a eukaryotic-type high molecular weight multi-functional enzyme (e.g., in wheat and maize) or a prokaryotic-type multi-subunit enzyme (e.g., in pea, soybean, tobacco and Arabidopsis). The other plant ACCase, located in the cytoplasm, is of the eukaryotic type. In Graminae, genes for both cytosolic and plastid eukaryotic-type ACCase are nuclear. No ACCase coding sequence can be found in the complete sequence of rice chloroplast DNA.
In other plants, subunits of ACCase other than the carboxyltransferase subunit encoded by a homolog of the E. coli accD gene, present in the chloroplast genome (Sasaki et al., 1995; Li and Cronan, 1992, Konishi et al., 1996), also is encoded in the nuclear DNA. Like the vast majority of plastid proteins, plastid ACCases are synthesized in the cytoplasm and then transported into the plastid. The amino acid sequence of the cytosolic and some subunits of the plastid ACCases from several plants have been deduced from genomic or cDNA sequences (e.g., Egli el al., 1995; Li and Cronan, 1992; Gornicki et al., 1994; Schulte et al., 1994; Shorrosh et al., 1994; Shorrosh et al., 1995; Roesler et al., 1994; Anderson et al., 1995). In plants, ACCase activity controls carbon flow through the fatty acid pathway and therefore may serve as an important regulation point of plant metabolism (Page et al., 1994; Post-Beitenmiller et al., 1992; Shintani and Ohlrogge, 1995).
The possibility of different ACCase isoforms, one present in plastids and another in the cytoplasm, is now accepted (Konishi et al., 1996). The rationale behind the existence of a cytoplasmic ACCase isoform is the requirement for malonyl-CoA in this cellular compartment, where it is used in fatty acid elongation and synthesis of secondary metabolites. Two isoforms of the multi-domain eukaryotic-type ACCase were found in maize, both consisting of &gt;200-kDa subunits but differing in size, herbicide sensitivity and immunological properties. The major form was found to be located in mesophyll chloroplasts. It is also the major ACCase in the endosperm and in embryos (Egli et al., 1993).
Many more genes and cDNAs encoding ACCases from various organisms have been cloned and sequenced. These sequences are available on Genbank.
1.2.2 Herbicide Resistance
Although the mechanisms of inhibition and resistance are unknown (Lichtenthaler, 1990), it has been shown that aryloxyphenoxypropionates and cyclohexane-1,3-dione derivatives, powerful herbicides effective against monocot weeds, inhibit fatty acid biosynthesis in sensitive plants.
The aryloxyphenoxypropionate class comprises derivatives of aryloxyphenoxy-propionic acid such as diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop and quizalofop. Several derivatives of cyclohexane-1,3-dione are also important post-emergence herbicides which also selectively inhibit monocot plants. This group comprises such compounds as oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim.
It is known that ACCase is the target enzyme for both of these classes of herbicide in Graminae monocots (grasses). Dicotyledonous plants, on the other hand, such as soybean, rape, sunflower, tobacco, canola, bean, tomato, potato, lettuce, spinach, carrot, alfalfa and cotton are resistant to these compounds, as are other eukaryotes and prokaryotes.
Important grain crops, such as wheat, rice, maize, barley, rye, and oats, however, are monocotyledonous plants, sensitive to these herbicides. Thus herbicides of the aryloxyphenoxypropionate and cyclohexane-1,3-dione groups are not useful in the agriculture of these important grain crops owing to the inactivation of monocot ACCase by such chemicals.
1.2.3 Deficiencies in the Prior Art
The genetic transformation of important commercial monocotyledonous agriculture crops with DNA segments encoding herbicide-resistant ACCase enzymes would be a revolution in the farming of such grains as wheat, rice, maize. barley, rye, and oats. Methods of identifying ACCase-encoding nucleic acid segments, and methods of identifying ACCase polypeptides resistant to herbicides would also be important in genetically engineering grain crops and the like with desirable herbicide-resistant qualities. Likewise the availability of DNA segments encoding monocotyledonous and dicotyledonous ACCase and nucleic acid segments derived therefrom would provide a much-needed means of genetically altering the activity of ACCase in vivo and in vitro.
What is lacking in the prior art, therefore, is the development of methods and processes for their use in creation of modified, transgenic plants which have altered herbicide resistance. Moreover, novel methods providing transgenic plants using DNA segments encoding ACCase polypeptides to modulate ACCase activity, fatty acid biosynthesis in general, and oil content of plant cells in specific, are greatly needed to provide transformed plants altered in such activity. Methods for determining ACCase activity in vivo and quantitating herbicide resistance in plants would also represent major improvements over the current state of the art.